CN107449916A

CN107449916A - Lung cancer and pulmonary nodule protein specificity spectrum and its construction method and application

Info

Publication number: CN107449916A
Application number: CN201710624070.8A
Authority: CN
Inventors: 陈宁; 李燕; 尚卓婷; 白晓婷; 徐敏; 伊娜; 林正宇
Original assignee: Beijing Banfi Biotechnology Co Ltd
Current assignee: Beijing Banfi Biotechnology Co Ltd
Priority date: 2017-07-27
Filing date: 2017-07-27
Publication date: 2017-12-08
Anticipated expiration: 2037-07-27
Also published as: CN107449916B

Abstract

The invention discloses a kind of lung cancer and pulmonary nodule protein specificity spectrum and its construction method and application.This method includes：Total protein, trypsin digestion, iTRAQ marks, two-dimensional liquid chromatography and tandem mass spectrum detection are extracted from the blood plasma of lung cancer/pulmonary nodule patient group and normal healthy controls person's group respectively, relative quantitative assay, obtains lung cancer/pulmonary nodule GAP-associated protein GAP characteristic spectrum；In lung cancer associated proteins characteristic spectrum, the albumen of differential expression is upregulated protein (SAA1, SERPINA1, CRP, IGHG4 and CFH) in patients with lung cancer group and normal healthy controls person's group；In pulmonary nodule GAP-associated protein GAP characteristic spectrum, the albumen of differential expression is upregulated protein (SERPINA1 and CRP) and down-regulation protein (SAA1, IGHG4 and CFH) in pulmonary nodule patient group and normal healthy controls person's group.The present invention helps to study lung cancer and pulmonary nodule biology essence, significant to lung cancer and pulmonary nodule specification parting.

Description

Lung cancer and pulmonary nodule protein specificity spectrum and its construction method and application

Technical field

The invention belongs to medical science and proteomics field, is related to a kind of lung cancer and pulmonary nodule protein specificity spectrum and its structure Construction method and application.

Background technology

Lung cancer is that morbidity and mortality growth is most fast, one of malignant tumour maximum to population health and life threat. Many countries all report that the morbidity and mortality of lung cancer substantially increase in the past 50 years, and male lung cancer morbidity and mortality are equal First of all malignant tumours is accounted for, the women incidence of disease accounts for second, and the death rate accounts for second.Pulmonary nodule be a kind of cause of disease not Bright lung's granulomatous diseases, domestic attention is extensively caused recently.

At present, the diagnostic method of lung cancer has x-ray inspection, bronchoscopy, cytolgical examination, exploratory thoracotomy, ECT inspections Look into, mediastinoscopy etc..But these are checked there is various drawbacks, or waste time and energy or considerable distress can be brought to patient.

Proteomics is to be proposed in 1994 by Williams and Wilkins, by finding specific protein, is Study of disease mechanism is given a clue.Comprehensive startup of plan, the research of proteomics are organized recently as human protein Also huge progress is generated, the quantitative approach applied in proteomics research mainly there are two kinds, and one kind is two-way based on tradition Quantifying in gel electrophoresis and dye-based, another is quantifying based on mass spectrum detection, including mark quantitative technique And two kinds of label-free technology (Label free quantification) (iTRAQ).ITRAQ combines two-dimentional gel chromatography It is mutually to be combined iTRAQ isotope labelling techniques, two-dimensional liquid chromatography technology, tandem mass spectrum technology with tandem mass spectrum, can be to institute There is protein to carry out isotope marks, during with mass spectrometry, signal ion shows as the peak of different mass-to-charge ratioes, according to the height of crest Degree and area can obtain accurate quantification of protein information, be adapted to the parallel analysis of multisample, available for finding more and disease Sick related protein.

SAA1：Serum amyloid A protein (serum amyloid A, SAA) is a kind of acute time limit reactive protein, is belonged to Heterogeneous proteinoid in apolipoprotein family, relative molecular weight about 12000.In the acute time limit reacts, through IL-1, IL-6 and TNF is stimulated, and SAA is synthesized in liver by the macrophage and fibroblast that are activated, can be increased to the 100- of initial concentration 1000 times, but half-life period is extremely short, only 50 minutes or so.Serum amyloid A protein is relevant with HDL (HDL), it The metabolism of HDL can be adjusted during inflammation.One especially important characteristic of serum amyloid A protein is its degraded Product can be deposited in different organs in a manner of amyloid A (AA) fibrillation, and this is one in chronic inflammatory diseases The serious complication of kind.

SERPINA1：α -1 antitrypsins (Alpha-1-antitrypsin, SERPINA1) are a kind of glycoprotein.Containing sugar 10%~20%, mainly synthesized, be distributed widely in normal human serum and body fluid by liver.It is most important albumen in serum Enzyme inhibitor, to fibrin ferment, urokinase etc., other enzymes also have inhibitory action；And a kind of Acute reaction protein, in inflammation Property illness when, α -1 antitrypsins can pass through capillary enter tissue fluid, inflammation locally it is often dense, to acute Inflammatory disease has certain limitations effect.

CRP：Mankind's c reactive protein (C-reactive protein, CRP) refers to be infected in body or tissue damage When blood plasma in some protein (acute protein) for steeply rising.CRP can with activating complement and strengthen phagocyte phagocytosis and Opsonic action is played, so as to remove the pathogenic microorganism of invasion body and damage, necrosis, the histocyte of apoptosis, in the day of body Important protective effect is played in right immunologic process.CRP concentration in Healthy Human Serum it is very low (<5mg/L), and in bacterium infection Or during tissue damage, its concentration significantly raises, therefore it is considered as its most worthy.

IGHG4：IgG4 is a hypotype in IgG, belongs to a kind of immunoglobulin, accounts for the 1~7% of IgG total amounts.IgG4 phases Closing property disease is a kind of and the closely related disease of IgG4 lymphocytes, and such disease is with the horizontal rise of serum IgG 4 and IgG4 Positive cell infiltrates a variety of organs and tissue and is characterized, and common afflicted organ includes lachrymal gland, pancreas and spatia retroperitonaeale etc., involves Organ or tissue anthorisma can be caused due to chronic inflammation and progression of fibrosis.

CFH：Complement factor H (Complement Factor H, CFH) is macrophage, Complement inhibition caused by blood platelet The factor, complement factor H are single-stranded structure, can be combined with C3b, suppress the C3 convertase of complement activation alternative pathway, and as benefit The confactor hydrolysis C3b of body FI.The generation of complement factor H assigns a kind of selective growth advantage of interior tumor cell, allows Cell evasion host immune system monitors, helps to avoid apoptosis in regulation and control in neoplastic cells escape human body.

The content of the invention

First purpose of the present invention is to provide a kind of construction method of lung cancer associated proteins characteristic spectrum.

The construction method of lung cancer associated proteins characteristic spectrum provided by the present invention is the method for non-diagnostic purpose, can specifically be wrapped Include following steps：Total protein is extracted in the Blood plasma in vitro organized respectively from patients with lung cancer group and normal healthy controls person, is entered with trypsase Row enzymolysis, iTRAQ marks are carried out to enzymolysis gained polypeptide, two-dimensional liquid chromatography and tandem mass spectrum detection are then carried out, to detection As a result relative quantitative assay is carried out, lung cancer associated proteins characteristic spectrum is obtained according to analysis result；

In the lung cancer associated proteins characteristic spectrum, differential expression in the patients with lung cancer group and normal healthy controls person's group Protein is the protein of up-regulated expression；The protein of the up-regulated expression is SAA1, SERPINA1, CRP, IGHG4 and CFH.

Second object of the present invention is to provide a kind of construction method of pulmonary nodule GAP-associated protein GAP characteristic spectrum.

The construction method of pulmonary nodule GAP-associated protein GAP characteristic spectrum provided by the present invention is the method for non-diagnostic purpose, specifically It may include following steps：Total protein is extracted from the Blood plasma in vitro of pulmonary nodule patient group and normal healthy controls person's group respectively, uses pancreas Protease is digested, and is carried out iTRAQ marks to enzymolysis gained polypeptide, is then carried out two-dimensional liquid chromatography and tandem mass spectrum inspection Survey, relative quantitative assay is carried out to testing result, pulmonary nodule GAP-associated protein GAP characteristic spectrum is obtained according to analysis result；

In the pulmonary nodule GAP-associated protein GAP characteristic spectrum, table in the pulmonary nodule patient group and normal healthy controls person's group Protein up to difference is the protein of up-regulated expression and the protein of expression downward；The protein of the up-regulated expression has SERPINA1 and CRP；The protein of the downward has SAA1, IGHG4 and CFH.

In the above described two methods, after total protein is extracted, it may also include the step of removing high-abundance proteins.Wherein, The high-abundance proteins are mainly albumin and IgG.In one embodiment of the invention, specifically using the more heavily adsorbs of Merck High-abundance proteins liquid-phase chromatographic column is gone to remove high-abundance proteins.

In the above described two methods, after iTRAQ marks are carried out to polypeptide obtained by enzymolysis, two-dimensional liquid chromatography is being carried out Before being detected with tandem mass spectrum, the step of C18 pillar desalting processings are carried out to sample may also include.Wherein, mobile phase is by flowing Dynamic phase A and Mobile phase B mix；The formula of mobile phase A is as follows：0.1% aqueous formic acid (% represents volumn concentration)； The formula of Mobile phase B is as follows：0.08% formic acid acetonitrile water (acetonitrile:Water=80:20, v/v, % represent volumn concentration).Point (% represents volumn concentration) following from gradient：

In the process, during the relative quantitative assay, using ProteinPilot softwares to the series connection matter Modal data carries out quantitative analysis, and the albumen for filtering out ratio >=1.5 is the albumen of up-regulated expression, and the albumen of ratio≤0.67 is table Up to the albumen of downward, the ratio is good for for the expressing quantity of the patients with lung cancer group or pulmonary nodule patient group with described The ratio of the expressing quantity of health collator group (reports that the peak area ratio of ion can represent same protein in second order mses Ratio of the same peptide fragment in different sample rooms).

The two-dimensional liquid chromatography is two-dimentional nanoliter liquid chromatogram, and actual conditions is as follows：The μ l of loading volume 8, on sandwich method Sample；Load flow rate pump 2 μ l/min, 15min；Mobile phase is to be mixed by mobile phase A and Mobile phase B；The formula of mobile phase A is such as Under：0.1% aqueous formic acid (% represents volumn concentration)；The formula of Mobile phase B is as follows：0.08% formic acid acetonitrile water (second Nitrile:Water=80:20, v/v, % represent volumn concentration)；The μ l/min of flow velocity 0.3 are separated, separation gradient is specific as follows：

The condition of the tandem mass spectrum is as follows：(1) source gas parameter：Ion injection electric：2.3kv；GS1 gas curtains：4； Curtain gas gas curtains：30 or 35；(2) flight time mass spectrum：Mass-to-charge ratio：350-1250；Accumulated time：0.25s；(3) from Son scanning：Mass-to-charge ratio：100-1500；Accumulated time：0.1s；Dynamic excludes the time：25s；Roll electronics：enabled；Adjust CE when using iTRAQ reagent：enabled；CES：5.

Third object of the present invention is to provide a kind of lung cancer associated proteins characteristic spectrum.

Lung cancer associated proteins characteristic spectrum provided by the present invention is the lung cancer associated proteins characteristic spectrum of non-diagnostic purpose, described The protein of differential expression is the protein of up-regulated expression in characteristic spectrum；The protein of the up-regulated expression be SAA1, SERPINA1, CRP, IGHG4 and CFH.

Fourth object of the present invention is to provide a kind of pulmonary nodule GAP-associated protein GAP characteristic spectrum.

Pulmonary nodule GAP-associated protein GAP characteristic spectrum provided by the present invention is special for the pulmonary nodule GAP-associated protein GAP of non-diagnostic purpose Sign is composed, the protein that the protein of differential expression is lowered for the protein of up-regulated expression with expression in the characteristic spectrum；The table Protein up to up-regulation has SERPINA1 and CRP；The protein of the downward has SAA1, IGHG4 and CFH.

The 5th purpose of the present invention is to provide a kind of application.

Application provided by the present invention is specially following (A) or (B)：

(A) it is used for material and the record for detecting SAA1, SERPINA1, CRP, IGHG4 and CFH this 5 kinds of expressing quantities There is application of the readable carrier of the description below in the kit for preparing auxiliary diagnosis lung cancer：

If compared with normal healthy controls person, the expression quantity of 5 kinds of albumen raises described in the Blood plasma in vitro sample of person under test, then The person under test is or candidate is patients with lung cancer；Conversely, then the person under test is not or candidate is not patients with lung cancer；

Be adjusted on described the ratio of the expressing quantity of the person under test and the expressing quantity of the normal healthy controls person >= 1.5。

(B) it is used for material and the record for detecting SAA1, SERPINA1, CRP, IGHG4 and CFH this 5 kinds of expressing quantities There is application of the readable carrier of the description below in the kit for preparing auxiliary diagnosis pulmonary nodule：

If compared with normal healthy controls person, this 2 hatching egg of the SERPINA1 and CRP described in the Blood plasma in vitro sample of person under test White expression quantity is raised, and the expression quantity of the SAA1, the IGHG4 and the CFH this 3 kinds of albumen is lowered, then described Person under test is or candidate is pulmonary nodule patient；Conversely, then the person under test is not or candidate is not pulmonary nodule patient；

Be adjusted on described the ratio of the expressing quantity of the person under test and the expressing quantity of the normal healthy controls person >= 1.5；Ratio≤0.67 of the expressing quantity of the person under test and the expressing quantity of the normal healthy controls person is adjusted under described.

The 6th purpose of the present invention is to provide a kind of system.

System provided by the present invention is following (C) or (D)：

(C) be used for auxiliary diagnosis lung cancer system, including for detect SAA1, SERPINA1, CRP, IGHG4 and CFH this 5 The material and data processing equipment of kind expressing quantity；Module 1 and module 2 are set in the data processing equipment, the module 1 has There is function shown in following (a1), the module 2 has function shown in following (a2)：

(a1) it is respectively compared the expression quantity for the 5 kinds of albumen being derived from the Blood plasma in vitro of person under test and normal healthy controls person；

(a2) according to the comparative result of (a1) according to being identified below whether the person under test is patients with lung cancer：If it is good for described Health collator compares, and the expression quantity of 5 kinds of albumen raises described in the Blood plasma in vitro sample of the person under test, then the person under test For or candidate be patients with lung cancer；Conversely, then the person under test is not or candidate is not patients with lung cancer；It is adjusted on described described to be measured Ratio >=1.5 of the expressing quantity of person and the expressing quantity of the normal healthy controls person.

(D) be used for auxiliary diagnosis pulmonary nodule system, including for detect SAA1, SERPINA1, CRP, IGHG4 and The material and data processing equipment of this 5 kinds of expressing quantities of CFH；Module 1 and module 2 are set in the data processing equipment, it is described Module 1 has function shown in following (a1), and the module 2 has function shown in following (a2)：

(a2) according to the comparative result of (a1) according to being identified below whether the person under test is pulmonary nodule patient：If with institute State normal healthy controls person to compare, the table of this 2 kinds of albumen of SERPINA1 and the CRP described in the Blood plasma in vitro sample of the person under test Raised up to amount, and the expression quantity of the SAA1, the IGHG4 and the CFH this 3 kinds of albumen is lowered, then the person under test For or candidate be pulmonary nodule patient；Conversely, then the person under test is not or candidate is not pulmonary nodule patient；It is adjusted on described Ratio >=1.5 of the expressing quantity of the person under test and the expressing quantity of the normal healthy controls person；It is adjusted under described described Ratio≤0.67 of the expressing quantity of person under test and the expressing quantity of the normal healthy controls person.

In the system, may also include at least one of following：(1) it is used to the progress total protein from Blood plasma in vitro take out Carry required reagent and/or instrument；(2) trypsase；(3) iTRAQ kits；(4) Two-dimensional Liquid Chromatography；(5) series connection matter Spectrometer；(6) it is used for the chromatographic column for removing high-abundance proteins；(7) the reverse silica gel absorption desalting columns of C18.

In the present invention, the SAA1 is that amino acid sequence is the protein shown in sequence 1 in sequence table；It is described SERPINA1 is that amino acid sequence is the protein shown in sequence 2 in sequence table；The CRP is that amino acid sequence is in sequence table Protein shown in sequence 3；The IGHG4 is that amino acid sequence is the protein shown in sequence 4 in sequence table；Described and CFH It is the protein in sequence table shown in sequence 5 for amino acid sequence.

The present invention marks quantitative protein group technology using iTRAQ, studies the differential protein of lung cancer and pulmonary nodule, most Find this 5 kinds of albumen of SAA1, SERPINA1, CRP, IGHG4 and CFH in the plasma sample of lung cancer and pulmonary nodule patient eventually Significant difference compared with normal healthy controls person be present in expression quantity.The present invention helps to study lung cancer and the biology sheet of pulmonary nodule Matter, to lung cancer and pulmonary nodule specification parting, it is significant to inquire into biology essence.

Brief description of the drawings

Fig. 1 is that this 5 kinds of characteristic protein matter of SAA1, SERPINA1, CRP, IGHG4, CFH are tied in normal person, lung cancer and lung Save the relative amount variation diagram in patient.Wherein, using the expression quantity of normal person as internal reference, other groups are all compared with normal person's group Compared with seeing the change of divergence multiple.

Embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.

SAA1 is that amino acid sequence is the protein shown in sequence 1 in sequence table；The SERPINA1 is amino acid sequence For the protein shown in sequence in sequence table 2；The CRP is that amino acid sequence is the protein shown in sequence 3 in sequence table；Institute It is that amino acid sequence is the protein shown in sequence 4 in sequence table to state IGHG4；Described and CFH is that amino acid sequence is sequence table Protein shown in middle sequence 5.

The structure of embodiment 1, lung cancer and pulmonary nodule protein specificity spectrum

First, sample and instrument

5 healthy adult person's plasma samples, pulmonary nodule patients blood plasma's sample of 5 untreateds, 5 underwent operative treatments Plasma of The Patients With Lung Cancer sample, the Plasma of The Patients With Lung Cancer sample of 5 untreateds.The person that respectively do not participate in the experiment is voluntary participation, its health And morbid state has laboratory and clinical diagnosis report to determine.All plasma samples are lower on an empty stomach in the morning to be extracted, and is used Disposal vacuum anticoagulant tube, peripheral blood 3.0ml is gathered, (3000r/min, 10min, 4 DEG C) is centrifuged in 4 hours, after drawing supernatant Each 50 μ l are distributed into, are stored in -80 DEG C of low temperature refrigerators.

It is as follows to test instrument equipment：The pillar for going high-abundance proteins of Merck ＆ Co., Inc.'s production；American AB Sciex is public Take charge of the iTRAQ kits of production；The C18 reverse phase silica gel adsorption desalination posts of waters companies production；American AB Sciex companies give birth to The mass spectrograph triple tof 5600systems of production；The ProteinPilot softwares (AB SCIEX) of American AB I companies production.

2nd, protein extraction

By the plasma sample that step 1 obtains after -80 DEG C of refrigerators take out, dissolved naturally at 4 DEG C, then use Merck ＆ Co., Inc. The pillar for going high-abundance proteins of production removes high-abundance proteins matter (high-abundance proteins mainly includes seralbumin, exempted from Epidemic disease Lysozyme), first peak is then collected, protein carries out the retention ultrafiltration removal elution of 3000Da molecular weight after flowing out pillar Salt in liquid, then (be formulated with Lysis lysates again in -20 DEG C of precipitates overnights, final protein precipitation with acetone：8.0M ureas, 50mM NH₄HCO₃, 1 × protease inhibitors) and dissolving.

3rd, the enzymolysis of albumen and iTRAQ marks

Protein example obtained by step 2 is subjected to Trypsin Induced, produced afterwards with American AB Sciex companies ITRAQ kits carry out sample labeling.Concrete operations are as follows：

1st, take 200 μ g protein solutions to be placed in centrifuge tube after quantification of protein, determined system to 125 μ l with 8M UA.

2nd, the 5 μ l 1M DTT solution now matched somebody with somebody are added into system, after mixing, 37 DEG C, 1h.

3rd, the 20 μ l 1M IAA solution now matched somebody with somebody are added into system, after mixing, lucifuge, react at room temperature 1h.

4th, all samples are drawn to be added in 10kD super filter tubes, 12000rpm (being no more than 14000 × g) centrifugation 20min, abandoned Fall collecting pipe bottom solution.

5th, 100 μ l UA are added into super filter tube, 12000rpm centrifugation 10min, are repeated 2 times.

6th, μ l, 12000rpm the centrifugation 20min of Dissolution Buffer 100 added in iTRAQ kits, are discarded Collecting pipe bottom solution, is repeated 3 times.

7th, the collecting pipe more renewed, trypsase is added in super filter tube, total amount 2-4 μ g are (with albumen quality than 1:50- 100), the μ l of volume 50,37 DEG C of reactions are overnight.

8th, next day, 12000rpm centrifugation 20min, peptide fragment solution centrifugal after enzymolysis, digestion is in collecting bottom of the tube.

9th, 50 μ l Dissolution Buffer are added into super filter tube again, centrifuges and merges with the filtrate of previous step.

10th, iTRAQ reagents are taken out from refrigerator, room temperature is equilibrated to, iTRAQ reagents is centrifuged to ttom of pipe；To every pipe iTRAQ 150 μ l organic solvents are added in reagent, and (4 mark reagents are ethanol, and 8 mark reagents are isopropanol, are ensured and organic phase after sample mixing Ratio be more than 70%), vortex oscillation, centrifuge to ttom of pipe.The present invention is specific as follows using 4 mark iTRAQ kits：114：It is strong Health collator's group；115：Untreated pulmonary nodule patient group；116：The patients with lung cancer group of underwent operative treatment；117：It is untreated Patients with lung cancer group.

11st, the half protein solution (i.e. 100 μ g) of enzymolysis sample is taken to be transferred in new centrifuge tube, remaining protein frozen Save backup；ITRAQ reagents are added in sample, vortex oscillation, centrifuge to ttom of pipe, react at room temperature 1h.Add 100 μ l water end Only react.

12nd, in order to detect labeling effciency and dosing accuracy, 1 μ l mixing is respectively taken out from 4 groups of samples, with Ziptip desalinations MALDI identifications are carried out afterwards, and confirmation flag reaction is good.

13rd, the sample after mixed mark, vortex oscillation, centrifuge to ttom of pipe；Vacuum refrigeration centrifugal drying；Sample after draining Freezen protective is stand-by.

4th, C18 pillars desalting processing

The C18 reverse phase silica gel adsorption desalination posts that will be produced through the mixing sample that above-mentioned steps three have marked with waters companies Desalting processing is carried out, the related salt component of the labelling kit brought into labeling process is removed, in order to subsequent analysis.Specific behaviour Make as follows：

1st, separated using 400 μ g polypeptide segments mixtures or E.coli protein extracts, detecting system situation.

2nd, the sample after step 3 mark is drained is redissolved with 150 μ l mobile phase As, vortex oscillation, 12000rpm centrifugations 20min, draw supernatant loading；Prepare 48 blank 1.5ml centrifuge tubes, it is isolated for collecting successively labeled as 1-48 Component 1-48；Flow velocity 0.8ml/min；Mobile phase is to be mixed by mobile phase A and Mobile phase B；The formula of mobile phase A is as follows： 0.1% aqueous formic acid (% represents volumn concentration)；The formula of Mobile phase B is as follows：0.08% formic acid acetonitrile water (acetonitrile: Water=80:20, v/v, % represent volumn concentration).It is following (% represents volumn concentration) to separate gradient：

3rd, since the 5th minute, eluate per minute is collected successively into 1-48 centrifuge tubes, according to peak intensity during appearance How much, merge, just more points of several pipes, appearance just collect a pipe to appearance for a long time less more.Vacuum refrigeration centrifugal drying；After draining Sample freezen protective it is stand-by.

5th, two-dimentional nanoliter liquid chromatographic detection and tandem mass spectrum detection

1st, according to ultraviolet monitoring situation, 48 components that step 4 is obtained merge into 10 components, use 30 μ during merging L " 2%ACN, 0.1%FA (% represent volumn concentration) ", adds first centrifuge tube, vortex oscillation and after centrifuging, and is transferred to Second centrifuge tube, until merging component, last is managed successively；Every group of μ l of sample 30 redissolves.

2nd, 12,000rpm, 10min, supernatant loading is drawn；It is general to draw 20 μ l.

3rd, the μ l of loading volume 8, take sandwich method loading.

4th, flow rate pump 2 μ l/min, 15min are loaded.

5th, mobile phase is to be mixed by mobile phase A and Mobile phase B；The formula of mobile phase A is as follows：0.1% formic acid is water-soluble Liquid (% represents volumn concentration)；The formula of Mobile phase B is as follows：0.08% formic acid acetonitrile water (acetonitrile:Water=80:20, v/ V, % represent volumn concentration).The μ l/min of flow velocity 0.3 are separated, separation gradient is slightly adjusted according to component 1-4 and 5-10, had Body gradient is following (% represents volumn concentration)：

6th, tandem mass spectrum detects

Every part of sample carries out matter after nano high performance liquid chromatography separations with the mass spectrographs of AB SCIEX TripleTOF 5600 Spectrum analysis.

Because iTRAQ reagents are equivalent, i.e., different isotopes, in first order Mass Spectrometer Method, divide after same polypeptide is marked Son amount is identical, with tandem mass spectrum method to being touched in first order Mass Spectrometer Method to precursor ion (precursor ion) Induction dissociation is hit, product ion is analyzed by second level mass spectrum.During second mass analysis, reporter group, quality Key fracture between balance group and polypeptides reactive group, mass balance group are lost, produce the report of low mass-to-charge ratio (m/z) from Son.Because second order mses can analyze the reporter group of relative molecular mass difference 1, the difference of different reporter group ionic strengths is just The relative abundance for the polypeptide that it is marked is represented, that is, the peak area ratio for reporting ion is exactly that the same peptide fragment of same protein exists The ratio of different sample rooms.Meanwhile the amido link fracture in polypeptide, a series of b ions and y ions are formed, obtain ion fragment Mass number, by data base querying and compare, corresponding protein precursor can be identified.

Mass spectrometry parameters set as follows：

(1) source gas parameter (being optimized according to different instrument states), it is below recommended value：

Ion spray voltage ion injection electrics：2.3kv

GS1 gas curtains：4；

Curtain gas gas curtains：30 or 35；

(2) TOF MS flight time mass spectrums：

M/z mass-to-charge ratioes：350-1250；

Accumulation time accumulated times：0.25s.

(3) Product ion scan ion scans：

M/z mass-to-charge ratioes：100-1500；

Accumulation time accumulated times：0.1s；

Dynamic exclusion time dynamics exclude the time：25s；

Rolling CE roll electronics：enabled；

Adjust CE when using iTRAQ reagent：enabled；

CES：5.

6th, proteinpilot softwares identification and analysis differential protein

Using the identification of proteinpilot softwares, analysis differential protein.It is specific as follows：The ms/ that deriving step five obtains Ms is composed, and mass spectral analysis initial data is RAW files, and with swissprot human databases, software Pro teinPilot (AB SCIEX checking storehouse identification and quantitative analysis) can be carried out to every kind of protein and (searches that storehouse parameter is as shown in table 1, and as a result filtration parameter is： Peptide FDR≤0.01), there is more than one peptide fragment and the albumen of 95% confidence level carries out data analysis, obtained by software Protein id, and protein ratio, and analyzing proteins are obtained according to mass spectrum peak area of the same protein in different groups The information such as matter biological function.

Table 1 searches storehouse parameter

7th, lung cancer and the characteristic protein matter of pulmonary nodule are selected

According to the characteristic protein matter for selecting lung cancer and pulmonary nodule as follows：Contrast healthy Normal group, untreated lung Portion's tubercle group and untreated lung cancer group, between carry out protein comparison, find out lung cancer and pulmonary nodule feature representation protein, Protein ratio shows its expression significantly up-regulation more than or equal to 1.5, and protein ratio shows its difference less than or equal to 0.67 The protein of expression is lowered.So-called ratio is the ratio compared with normal healthy controls person.

8th, result

As a result show：5 kinds of characteristic protein matter of lung cancer and pulmonary nodule are obtained through above-mentioned detection and analysis, respectively SAA1, SERPINA1, CRP, IGHG4 and CFH.The confidence level of this 5 kinds of albumen is equivalent and unused protscor values are as shown in table 2. This ratio of 5 kinds of characteristic proteins between each group is as shown in table 3 in lung cancer and pulmonary nodule.SAA1、SERPINA1、CRP、 Relative amount variation diagram such as Fig. 1 institute of this 5 kinds of characteristic protein matter of IGHG4, CFH in normal person, lung cancer and pulmonary nodule patient Show.

The confidence level equivalence and unused protscor values of 5 kinds of feature serum proteins of the lung cancer of table 2 and pulmonary nodule

Ratio of the 5 kinds of characteristic serum proteins of the lung cancer of table 3 and pulmonary nodule between each group

Note：114：Normal control；115：Lung's section tissue is not treated；116：Lung cancer group operative treatment；117：Lung cancer group is not Treatment.

As seen from Table 3, compared with healthy Normal group, SAA1 in the plasma sample of the patients with lung cancer group of untreated, This 5 kinds of albumen of SERPINA1, CRP, IGHG4 and CFH show as up-regulation and (are more than with the protein ratio of healthy Normal group Equal to 1.5).Compared with healthy Normal group, in the plasma sample of the pulmonary nodule patient of untreated group SERPINA1 and This 2 kinds of albumen of CRP show as up-regulation (being more than or equal to 1.5 with the protein ratio of healthy Normal group)；SAA1、IGHG4 This 3 kinds of albumen show as lowering and (being less than or equal to 0.67 with the protein ratio of healthy Normal group) with CFH.

<110>Beijing Bang Fei bio tech ltd

<120>Lung cancer and pulmonary nodule protein specificity spectrum and its construction method and application

<130> GNCLN171508

<160> 5

<170> PatentIn version 3.5

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35 40 45

Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln

50 55 60

Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser

65 70 75 80

Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr

85 90 95

His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro

100 105 110

Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn

115 120 125

Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu

130 135 140

Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys

145 150 155 160

Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu

165 170 175

Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys

180 185 190

Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu

195 200 205

Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val

210 215 220

Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val

225 230 235 240

Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys

245 250 255

Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala

260 265 270

Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu

275 280 285

Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp

290 295 300

Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr

305 310 315 320

Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe

325 330 335

Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys

340 345 350

Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly

355 360 365

Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile

370 375 380

Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu

385 390 395 400

Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr

405 410 415

Gln Lys

<210> 3

<211> 224

<212> PRT

<213>People（Homo sapiens）

<400> 3

Met Glu Lys Leu Leu Cys Phe Leu Val Leu Thr Ser Leu Ser His Ala

1 5 10 15

Phe Gly Gln Thr Asp Met Ser Arg Lys Ala Phe Val Phe Pro Lys Glu

20 25 30

Ser Asp Thr Ser Tyr Val Ser Leu Lys Ala Pro Leu Thr Lys Pro Leu

35 40 45

Lys Ala Phe Thr Val Cys Leu His Phe Tyr Thr Glu Leu Ser Ser Thr

50 55 60

Arg Gly Tyr Ser Ile Phe Ser Tyr Ala Thr Lys Arg Gln Asp Asn Glu

65 70 75 80

Ile Leu Ile Phe Trp Ser Lys Asp Ile Gly Tyr Ser Phe Thr Val Gly

85 90 95

Gly Ser Glu Ile Leu Phe Glu Val Pro Glu Val Thr Val Ala Pro Val

100 105 110

His Ile Cys Thr Ser Trp Glu Ser Ala Ser Gly Ile Val Glu Phe Trp

115 120 125

Val Asp Gly Lys Pro Arg Val Arg Lys Ser Leu Lys Lys Gly Tyr Thr

130 135 140

Val Gly Ala Glu Ala Ser Ile Ile Leu Gly Gln Glu Gln Asp Ser Phe

145 150 155 160

Gly Gly Asn Phe Glu Gly Ser Gln Ser Leu Val Gly Asp Ile Gly Asn

165 170 175

Val Asn Met Trp Asp Phe Val Leu Ser Pro Asp Glu Ile Asn Thr Ile

180 185 190

Tyr Leu Gly Gly Pro Phe Ser Pro Asn Val Leu Asn Trp Arg Ala Leu

195 200 205

Lys Tyr Glu Val Gln Gly Glu Val Phe Thr Lys Pro Gln Leu Trp Pro

210 215 220

<210> 4

<211> 327

<212> PRT

<213>People（Homo sapiens）

<400> 4

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 5

<211> 1231

<212> PRT

<213>People（Homo sapiens）

<400> 5

Met Arg Leu Leu Ala Lys Ile Ile Cys Leu Met Leu Trp Ala Ile Cys

1 5 10 15

Val Ala Glu Asp Cys Asn Glu Leu Pro Pro Arg Arg Asn Thr Glu Ile

20 25 30

Leu Thr Gly Ser Trp Ser Asp Gln Thr Tyr Pro Glu Gly Thr Gln Ala

35 40 45

Ile Tyr Lys Cys Arg Pro Gly Tyr Arg Ser Leu Gly Asn Val Ile Met

50 55 60

Val Cys Arg Lys Gly Glu Trp Val Ala Leu Asn Pro Leu Arg Lys Cys

65 70 75 80

Gln Lys Arg Pro Cys Gly His Pro Gly Asp Thr Pro Phe Gly Thr Phe

85 90 95

Thr Leu Thr Gly Gly Asn Val Phe Glu Tyr Gly Val Lys Ala Val Tyr

100 105 110

Thr Cys Asn Glu Gly Tyr Gln Leu Leu Gly Glu Ile Asn Tyr Arg Glu

115 120 125

Cys Asp Thr Asp Gly Trp Thr Asn Asp Ile Pro Ile Cys Glu Val Val

130 135 140

Lys Cys Leu Pro Val Thr Ala Pro Glu Asn Gly Lys Ile Val Ser Ser

145 150 155 160

Ala Met Glu Pro Asp Arg Glu Tyr His Phe Gly Gln Ala Val Arg Phe

165 170 175

Val Cys Asn Ser Gly Tyr Lys Ile Glu Gly Asp Glu Glu Met His Cys

180 185 190

Ser Asp Asp Gly Phe Trp Ser Lys Glu Lys Pro Lys Cys Val Glu Ile

195 200 205

Ser Cys Lys Ser Pro Asp Val Ile Asn Gly Ser Pro Ile Ser Gln Lys

210 215 220

Ile Ile Tyr Lys Glu Asn Glu Arg Phe Gln Tyr Lys Cys Asn Met Gly

225 230 235 240

Tyr Glu Tyr Ser Glu Arg Gly Asp Ala Val Cys Thr Glu Ser Gly Trp

245 250 255

Arg Pro Leu Pro Ser Cys Glu Glu Lys Ser Cys Asp Asn Pro Tyr Ile

260 265 270

Pro Asn Gly Asp Tyr Ser Pro Leu Arg Ile Lys His Arg Thr Gly Asp

275 280 285

Glu Ile Thr Tyr Gln Cys Arg Asn Gly Phe Tyr Pro Ala Thr Arg Gly

290 295 300

Asn Thr Ala Lys Cys Thr Ser Thr Gly Trp Ile Pro Ala Pro Arg Cys

305 310 315 320

Thr Leu Lys Pro Cys Asp Tyr Pro Asp Ile Lys His Gly Gly Leu Tyr

325 330 335

His Glu Asn Met Arg Arg Pro Tyr Phe Pro Val Ala Val Gly Lys Tyr

340 345 350

Tyr Ser Tyr Tyr Cys Asp Glu His Phe Glu Thr Pro Ser Gly Ser Tyr

355 360 365

Trp Asp His Ile His Cys Thr Gln Asp Gly Trp Ser Pro Ala Val Pro

370 375 380

Cys Leu Arg Lys Cys Tyr Phe Pro Tyr Leu Glu Asn Gly Tyr Asn Gln

385 390 395 400

Asn Tyr Gly Arg Lys Phe Val Gln Gly Lys Ser Ile Asp Val Ala Cys

405 410 415

His Pro Gly Tyr Ala Leu Pro Lys Ala Gln Thr Thr Val Thr Cys Met

420 425 430

Glu Asn Gly Trp Ser Pro Thr Pro Arg Cys Ile Arg Val Lys Thr Cys

435 440 445

Ser Lys Ser Ser Ile Asp Ile Glu Asn Gly Phe Ile Ser Glu Ser Gln

450 455 460

Tyr Thr Tyr Ala Leu Lys Glu Lys Ala Lys Tyr Gln Cys Lys Leu Gly

465 470 475 480

Tyr Val Thr Ala Asp Gly Glu Thr Ser Gly Ser Ile Thr Cys Gly Lys

485 490 495

Asp Gly Trp Ser Ala Gln Pro Thr Cys Ile Lys Ser Cys Asp Ile Pro

500 505 510

Val Phe Met Asn Ala Arg Thr Lys Asn Asp Phe Thr Trp Phe Lys Leu

515 520 525

Asn Asp Thr Leu Asp Tyr Glu Cys His Asp Gly Tyr Glu Ser Asn Thr

530 535 540

Gly Ser Thr Thr Gly Ser Ile Val Cys Gly Tyr Asn Gly Trp Ser Asp

545 550 555 560

Leu Pro Ile Cys Tyr Glu Arg Glu Cys Glu Leu Pro Lys Ile Asp Val

565 570 575

His Leu Val Pro Asp Arg Lys Lys Asp Gln Tyr Lys Val Gly Glu Val

580 585 590

Leu Lys Phe Ser Cys Lys Pro Gly Phe Thr Ile Val Gly Pro Asn Ser

595 600 605

Val Gln Cys Tyr His Phe Gly Leu Ser Pro Asp Leu Pro Ile Cys Lys

610 615 620

Glu Gln Val Gln Ser Cys Gly Pro Pro Pro Glu Leu Leu Asn Gly Asn

625 630 635 640

Val Lys Glu Lys Thr Lys Glu Glu Tyr Gly His Ser Glu Val Val Glu

645 650 655

Tyr Tyr Cys Asn Pro Arg Phe Leu Met Lys Gly Pro Asn Lys Ile Gln

660 665 670

Cys Val Asp Gly Glu Trp Thr Thr Leu Pro Val Cys Ile Val Glu Glu

675 680 685

Ser Thr Cys Gly Asp Ile Pro Glu Leu Glu His Gly Trp Ala Gln Leu

690 695 700

Ser Ser Pro Pro Tyr Tyr Tyr Gly Asp Ser Val Glu Phe Asn Cys Ser

705 710 715 720

Glu Ser Phe Thr Met Ile Gly His Arg Ser Ile Thr Cys Ile His Gly

725 730 735

Val Trp Thr Gln Leu Pro Gln Cys Val Ala Ile Asp Lys Leu Lys Lys

740 745 750

Cys Lys Ser Ser Asn Leu Ile Ile Leu Glu Glu His Leu Lys Asn Lys

755 760 765

Lys Glu Phe Asp His Asn Ser Asn Ile Arg Tyr Arg Cys Arg Gly Lys

770 775 780

Glu Gly Trp Ile His Thr Val Cys Ile Asn Gly Arg Trp Asp Pro Glu

785 790 795 800

Val Asn Cys Ser Met Ala Gln Ile Gln Leu Cys Pro Pro Pro Pro Gln

805 810 815

Ile Pro Asn Ser His Asn Met Thr Thr Thr Leu Asn Tyr Arg Asp Gly

820 825 830

Glu Lys Val Ser Val Leu Cys Gln Glu Asn Tyr Leu Ile Gln Glu Gly

835 840 845

Glu Glu Ile Thr Cys Lys Asp Gly Arg Trp Gln Ser Ile Pro Leu Cys

850 855 860

Val Glu Lys Ile Pro Cys Ser Gln Pro Pro Gln Ile Glu His Gly Thr

865 870 875 880

Ile Asn Ser Ser Arg Ser Ser Gln Glu Ser Tyr Ala His Gly Thr Lys

885 890 895

Leu Ser Tyr Thr Cys Glu Gly Gly Phe Arg Ile Ser Glu Glu Asn Glu

900 905 910

Thr Thr Cys Tyr Met Gly Lys Trp Ser Ser Pro Pro Gln Cys Glu Gly

915 920 925

Leu Pro Cys Lys Ser Pro Pro Glu Ile Ser His Gly Val Val Ala His

930 935 940

Met Ser Asp Ser Tyr Gln Tyr Gly Glu Glu Val Thr Tyr Lys Cys Phe

945 950 955 960

Glu Gly Phe Gly Ile Asp Gly Pro Ala Ile Ala Lys Cys Leu Gly Glu

965 970 975

Lys Trp Ser His Pro Pro Ser Cys Ile Lys Thr Asp Cys Leu Ser Leu

980 985 990

Pro Ser Phe Glu Asn Ala Ile Pro Met Gly Glu Lys Lys Asp Val Tyr

995 1000 1005

Lys Ala Gly Glu Gln Val Thr Tyr Thr Cys Ala Thr Tyr Tyr Lys

1010 1015 1020

Met Asp Gly Ala Ser Asn Val Thr Cys Ile Asn Ser Arg Trp Thr

1025 1030 1035

Gly Arg Pro Thr Cys Arg Asp Thr Ser Cys Val Asn Pro Pro Thr

1040 1045 1050

Val Gln Asn Ala Tyr Ile Val Ser Arg Gln Met Ser Lys Tyr Pro

1055 1060 1065

Ser Gly Glu Arg Val Arg Tyr Gln Cys Arg Ser Pro Tyr Glu Met

1070 1075 1080

Phe Gly Asp Glu Glu Val Met Cys Leu Asn Gly Asn Trp Thr Glu

1085 1090 1095

Pro Pro Gln Cys Lys Asp Ser Thr Gly Lys Cys Gly Pro Pro Pro

1100 1105 1110

Pro Ile Asp Asn Gly Asp Ile Thr Ser Phe Pro Leu Ser Val Tyr

1115 1120 1125

Ala Pro Ala Ser Ser Val Glu Tyr Gln Cys Gln Asn Leu Tyr Gln

1130 1135 1140

Leu Glu Gly Asn Lys Arg Ile Thr Cys Arg Asn Gly Gln Trp Ser

1145 1150 1155

Glu Pro Pro Lys Cys Leu His Pro Cys Val Ile Ser Arg Glu Ile

1160 1165 1170

Met Glu Asn Tyr Asn Ile Ala Leu Arg Trp Thr Ala Lys Gln Lys

1175 1180 1185

Leu Tyr Ser Arg Thr Gly Glu Ser Val Glu Phe Val Cys Lys Arg

1190 1195 1200

Gly Tyr Arg Leu Ser Ser Arg Ser His Thr Leu Arg Thr Thr Cys

1205 1210 1215

Trp Asp Gly Lys Leu Glu Tyr Pro Thr Cys Ala Lys Arg

1220 1225 1230

Claims

1. a kind of construction method of lung cancer associated proteins characteristic spectrum, comprises the following steps：It is right from patients with lung cancer group and health respectively According to total protein is extracted in the Blood plasma in vitro of person's group, digested with trypsase, iTRAQ marks carried out to enzymolysis gained polypeptide, Then two-dimensional liquid chromatography and tandem mass spectrum detection are carried out, relative quantitative assay is carried out to testing result, obtained according to analysis result Obtain lung cancer associated proteins characteristic spectrum；

In the lung cancer associated proteins characteristic spectrum, the albumen of differential expression in the patients with lung cancer group and normal healthy controls person's group Matter is the protein of up-regulated expression；The protein of the up-regulated expression is SAA1, SERPINA1, CRP, IGHG4 and CFH.

2. a kind of construction method of pulmonary nodule GAP-associated protein GAP characteristic spectrum, comprises the following steps：Respectively from pulmonary nodule patient's group Total protein is extracted with the Blood plasma in vitro of normal healthy controls person's group, is digested with trypsase, enzymolysis gained polypeptide is carried out ITRAQ is marked, and then carries out two-dimensional liquid chromatography and tandem mass spectrum detection, and relative quantitative assay is carried out to testing result, according to Analysis result obtains pulmonary nodule GAP-associated protein GAP characteristic spectrum；

In the pulmonary nodule GAP-associated protein GAP characteristic spectrum, expression is poor in the pulmonary nodule patient group and normal healthy controls person's group Different protein is the protein of up-regulated expression and the protein of expression downward；The protein of the up-regulated expression has SERPINA1 And CRP；The protein of the downward has SAA1, IGHG4 and CFH.

3. method according to claim 1 or 2, it is characterised in that：During the relative quantitative assay, use ProteinPilot softwares carry out quantitative analysis to the tandem mass spectrum data, and the albumen and expression for filtering out up-regulated expression are lowered Protein value.

A kind of 4. lung cancer associated proteins characteristic spectrum, it is characterised in that：The protein of differential expression is in expression in the characteristic spectrum The protein of tune；The protein of the up-regulated expression is SAA1, SERPINA1, CRP, IGHG4 and CFH.

A kind of 5. pulmonary nodule GAP-associated protein GAP characteristic spectrum, it is characterised in that：The protein of differential expression is table in the characteristic spectrum The protein lowered up to the protein of up-regulation and expression；The protein of the up-regulated expression has SERPINA1 and CRP；The downward Protein have SAA1, IGHG4 and CFH.

6. for detecting the material of SAA1, SERPINA1, CRP, IGHG4 and CFH this 5 kinds of expressing quantities and recording following Application of the readable carrier of content in the kit for preparing auxiliary diagnosis lung cancer：

If compared with normal healthy controls person, the expression quantity of 5 kinds of albumen raises described in the Blood plasma in vitro sample of person under test, then described Person under test is or candidate is patients with lung cancer；Conversely, then the person under test is not or candidate is not patients with lung cancer.

7. for detecting the material of SAA1, SERPINA1, CRP, IGHG4 and CFH this 5 kinds of expressing quantities and recording following Application of the readable carrier of content in the kit for preparing auxiliary diagnosis pulmonary nodule：

If compared with normal healthy controls person, this 2 kinds of albumen of the SERPINA1's and CRP described in the Blood plasma in vitro sample of person under test Expression quantity is raised, and the expression quantity of the SAA1, the IGHG4 and the CFH this 3 kinds of albumen is lowered, then described to be measured Person is or candidate is pulmonary nodule patient；Conversely, then the person under test is not or candidate is not pulmonary nodule patient.

8. for the system of auxiliary diagnosis lung cancer, including for detecting SAA1, SERPINA1, CRP, IGHG4 and CFH this 5 hatching egg The material and data processing equipment of white expression quantity；Module 1 and module 2 are set in the data processing equipment, the module 1 has such as Under function shown in (a1), the module 2 has function shown in following (a2)：

(a2) according to the comparative result of (a1) according to being identified below whether the person under test is patients with lung cancer：It is if right with the health Compared according to person, the expression quantity of 5 kinds of albumen raises described in the Blood plasma in vitro sample of the person under test, then the person under test be or Candidate is patients with lung cancer；Conversely, then the person under test is not or candidate is not patients with lung cancer.

9. for auxiliary diagnosis pulmonary nodule system, including for detect SAA1, SERPINA1, CRP, IGHG4 and CFH this 5 The material and data processing equipment of kind expressing quantity；Module 1 and module 2 are set in the data processing equipment, the module 1 has There is function shown in following (a1), the module 2 has function shown in following (a2)：

(a2) according to the comparative result of (a1) according to being identified below whether the person under test is pulmonary nodule patient：If it is good for described Health collator compares, the expression quantity of this 2 kinds of albumen of SERPINA1 and the CRP described in the Blood plasma in vitro sample of the person under test Raise, and the expression quantity of the SAA1, the IGHG4 and the CFH this 3 kinds of albumen is lowered, then the person under test be or Candidate is pulmonary nodule patient；Conversely, then the person under test is not or candidate is not pulmonary nodule patient.

10. system according to claim 8 or claim 9, it is characterised in that：At least one in also including as follows in the system Kind：

(1) it is used to carry out the reagent and/or instrument needed for total protein extracting from Blood plasma in vitro；

(2) trypsase；

(3) iTRAQ kits；

(4) Two-dimensional Liquid Chromatography；

(5) tandem mass spectrometer；

(6) it is used for the chromatographic column for removing high-abundance proteins；

(7) the reverse silica gel absorption desalting columns of C18.